11.1-11.7 DNA replication, translation Flashcards
intergenic space
noncoding DNA
human has 3.2 billion BPs, 21,000 genes, and long stretches of intergenic regions (noncoding DNA) which may direct chromatin structure / regulate genes / no known function
include TANDEM REPEATS and TRANSPOSONS
gene
codes for gene product
includes REGULATORY sites (promoters, transcription stop sites), and “ACTIVE” site (codes for protein or non-coding RNA)
single nucleotide polymorphism
ALLELE - 1 nucleotide change once every 1000 bp’s, “snips” are essentially mutations
- > sickle-cell anemia, beta-thalassemia, cystic fibrosis
- > see picture
copy number variation
structural variations in genome (large regions of genome 10^3-10^6 can be duplicated)
DUPLICATION OF DNA, or DELETED
5-10% of human genome
Tandem repeats
short sequence of nucleotides repeat right after each other, 3-100 times
Transposons
genes code for tranposase (“cut and paste” activity)
“jumping” around the genome; very mischievous
CAUSES or REVERSES mutations
Hershey and Chase
proved DNA is the genetic material
Watch video
ribosome
very large, rRNA and protein
UGA
university of georgia at atlanta (stop codon)
topoisomerase
cuts strand and unwraps helix, releasing excess TENSION
single-strand binding protein
proteins DNA that has been de-doubled-stranded (much less stable), leads to the OPEN COMPLEX (ready to begin replication)
Initiation-open complex
an RNA primer MUST be synthesized for each template strand – primosome (contains RNA polymerase primase)
DNA pol CANNOT START a DNA chain from scratch. The RNA primer is 8-12 nucleotides long, later replaced by DNA
DNA Pol
part of a larger complex of proteins called the REPLISOME
prokaryote - 13 components
eukaryote - 27 proteins
thermo of DNA replication
removal and hydrolysis of pryophosphate (two phosphates) from each dTNP (dTNP is the nucleotide with 3 phosphates)
Okazaki fragments are joined by
DNA ligase
prokaryotic DNA polymerases (III and I)
I, II, III, and IV
III - elongation of leading strand; has 5-3 polymerase and 3-5 exonuclease activity; FAST
I - adds nucleotides at the RNA primer, 5-3 polymerase activity, only adds 15-20 nucleotides a second, taken over by pol III 400 bps downstream; capable of 3-5 exonuclease activity; removes the primer via 5-3 exonuclease activity; important for excision repair
II - backup for III
IV and V - error-prone in 5-3 polymerase activity , function to stall other polymerase enzymes at replication forks
telomerase
RIBONUCLEOPROTEIN: adds repetitive nucleotides to ends of chromosomes
expressed only in germ line, stem cells, and some white blood cells
transition/transversion
substitute pyrimidines for a pyrimidine, purine for a purine
transversion is more severe
translocation
common in cancers
two regions swap locations
occurs in genetic recombination
produces a new gene product
loss of heterozygosity
one allele of a certain gene is lost, due to deletion or recombination
hemizygous - only 1 gene copy.
direct reversal of dna repair
bacteria and plants can repair UV-induced pyrimidine photodimers DIRECTLY
nucleotide excision repair is the next step
main mechanism of repair in humans, but can introduce a mutation
homology-dependency repair
DNA is redundant (two copies)
excision repair (before DNA replication) and post-replication repair (after DNA replication)
excision repair
BEFORE dna replication; removes defective base and replaces them
post-replication repair
MMR - mismatch repair pathway - targets mistakes not repaired by DNA polymerase proofreading
DURING dna replication; methylation helps determine which is wrong (A or G)
during replication, the free 3’ end and Okazaki fragments are found
homologous recombination to repair DS breaks (see illustration p. 222)
nuclease and helicase generate single-stranded DNA
they find complementary sister chromatid
dna pol and ligase build new DNA
non-homologous recombination repair of DS break
not perfect
Open-reading frame (ORF)
after the 5-3 UTR (untranslated region - initiation and regulation), ORF is start to stop codon (coding region)
monocistronic v. polycistronic
“one mRNA, one protein” - eukaryotes
polycistronic - one mRNA, multiple proteins - prokaryotes; translation can start in multiple places! termination and initiation sequences between each ORF
NOTE; PROKARYOTES DON’T PROCESS mRNA, only eukaryotes have hnRNA (which are modified: addition of cap and tail, splicing)
rRNA (4 types)
18S, 5.8S, 28S, and 5S
serve in ribosome - along with polypeptide chains (proteins), 1 rRNA provides catalytic function of the ribosome (a RIBOZYME)
Driving force of replication/transcription
removal and hydrolysis of pyrophosphate from each nucleotide added
promotor (transcription) versus start site
the area of DNA that ACTIVATES transcription (origin is where DNA is replicated)
START site is called the “START SITE”
antisense and sense strands
the DNA template is the antisense (because it’s the ANTI of the mRNA)
coding strand (“sense”); +1 is the first nucleotide transcribed
mRNA grows on the 3’ end -> proceeds downstream
RNA polymerase
pribnow box (-10) and -35 sequence forms a CLOSED COMPLEX
RNA pol must unwind DNA
RNA pol HOLOenzyme bound at promotor with a region of single-stranded DNA is termed the OPEN COMPLEX (difference is that the DNA is unwound)
bacteria transcription
All RNA is made of the same RNA pol
a large enzyme - 5 subunits
Core enzyme - rapid elongation
Subunit sigma factor (forms the HOLO-enzyme) - responsible for initiation
Initiation -> elongation -> termination
sigma factor
helps prokaryotes find promoters
processive elogation
core enzyme moves along the DNA downstream, a bubble forms when the DNA is unwound
rho helps determine when to end, polymerase falls off and bubble is closed
eukaryotes location of transcription
nucleus
modify in the nucleus, transport to cytoplasm where it is translated
prokaryotes translate…
…while transcribing, no post-processing involved
eukaryotic splicing
extensive modification after transcription - splicing
these non-coding regions (introns) may code for proteins, contain ENHANCERS or REGULATORY sequences
spliceosome (and snRNA)
mediates eukaryotic mRNA splicing
100 proteins and 5 small nuclear RNA molecules
the snRNA “detects” the intron (see image p. 227)
ALTERNATIVE splicing - multiple ways of splicing hnRNA
cap and tails must be added (5’ cap, and 3’ poly-A tail)
RNA polymerases
I - rRNa
II - hnRNA, most snRNA
III - tRNA
tRNA, the protein bonds to
the 3’ end
wobble hypothesis
of anticodon: 3rd position is more flexible than 1 and 2
5’ base anticodon (tRNA) - 3’ base in codon (mRNA)
G -> C or U
U -> A or G
I -> A, U, C
Amino Acid Activation p. 231
Need help understanding this…
- amino acid attaches to AMP to form aminoacyl AMP
- pyrophosphate leaving group is hydrolyzed to 2 orthophosphates (delta_G «_space;0)
- tRNA loading (deltaG»_space; 0) - driven by the destruction of the aminoacyl-AMP bond (basically AMP is released fro aminoacyl amp, leading to the aminoacyl-tRNA)
Final product: Aminoacyl-tRNA
Requires: 2 atp equivalents
aminoacyl-tRNA synthetase anzyme
family of enzymes that recognizes the tRNA and amino acid , puts them together – HIGHLY SPECIFIC
prokaryotic ribosome
30S and 50S => 70S
30S -> 16S rRNA and 21 peptides
50S -> 23S and 5S and 31 peptides
eukaryotic ribosome
80S ribosome
large -> 60S -> 5S, 5.8S, 28S and 46 peptides
small -> 40S -> 18S, 33 peptides
the 5’ end ORF
upstream regulatory sequence is essential for initiation, we have a ribosome binding site (Shine-Dalgarno sequence) located at -10. SD sequence is complementary to a pyrimidine rich region on the small subunit
translation has three distinct stages
initiation, elongation, termination
initiation
30S binds two initiation proteins called IF1 and IF3, which binds to the mRNA transcript, then aminoacyl-tRNA joins, then IF2, which is bound to 1 GTP.
50S completes the complex
Powered by hydrolysis of 1 GTP molecule
first aminoacyl-tRNA is called the initiator tRNA (fMet-tRNA) - sits in P site of 70S ribosome, hydrogen-bonded with the start codon
elongation
three-steps
- second aminoacyl-tRNA enters the A site, h-bonds with the second codon (1 GTP)
- peptidyl transferase activity of large ribosomal subunit (23S) catalyzes formation of a peptide bond between fMet and the second amino acid (driven by hydrolysis of tRNA and amino acid p. 231)
- translocation - tRNA #1 moves to E site, tRNA #2 (holding the growing peptide) moves to the P site, and next codon moves into the A site. (1 GTP)
termination
when stop codon appears in the A site
a release factor enters the A site, causes peptidyl transferase to hydrolyze the bond between the last tRNA and completed polypeptide
5’ UTR
untranslated region - common in eukaryotes (e.g. Kozak sequence), located several nucleotides before start codon
eukaryotic transcription begins…
…with formation of initiation complex
43S small ribosomal subunit forms + Met-tRNA_Met + several proteins called eukaryotic initiation factors (eIFs);
this is recruited to 5’ capped end of transcript, by initiation complex of proteins; additional proteins recruited (polyA tail binding protein)
Initiation complex starts scanning the 5’ end, looking for a start codon
Once found, the large ribosomal subunit (60S) is recruited and translation begins
eukaryotes have *** elongation factors
2
cap-dependent translation
the mRNA cap, 5’ end, is so important for translation
but cap-independent translation was discovered
site has IRES (ribosome entry site) -> helps apoptosis or deal with stress
